Disclosed herein are methods of forming a fiber mat, involving forming an aqueous solution of at least one protein, at least one polysaccharide, and optionally a plasticizer, and electrospinning the aqueous solution onto a collector to form a mat.
Casein represents approximately 80% of the protein content in milk. It is composed of alpha s1, alpha s2, beta- and kappa-casein in the ratios of approximately 40:10:35:12 (Fox, P. F., The milk protein system, In: Developments in Dairy Chemistry—4, Functional Milk Proteins, P. F. Fox, ed., Elsevier Applied Science, New York, 1989) and exists in a colloidal complex bound together by Ca—P linkages and hydrophobic interactions. Kappa-casein stabilizes the exterior of the micelle, preventing precipitation of the other caseins through hydrophilic interactions. Because casein is a phosphoprotein, it binds Ca in proportion to the number of P groups and may also bind other minerals such as Zn. Because of its unique open structure, the casein micelle structure as it exists in milk has been proposed as a nano encapsulant for targeted nutritional or drug delivery (Livney, Y. D., et al., Nanoencapsulation of hydrophobic nutraceutical substances within casein micelles, XIVth International Workshop on Bioencapsulation, Lausanne, CH. 07-4 pg. 1-4 (2006)).
Casein has very low amounts of cysteine and no disulfide linkages, giving it a random coil structure (Gennadios, A., et al., Chapter 9, Edible Coatings and Films Based on Proteins In Edible Coatings and Films to Improve Food Quality, J. M. Krochta et al. eds., Technomic Publishing Co., Inc., Lancaster, Pa., 1994) with very little secondary or tertiary structure. The random coil structure is responsible for the ability of casein to form films. The large number of proline residues allows bending of the protein chains but prevents building of secondary structures. Casein is very sensitive to pH which dictates its structure in solution, and ultimately its function. At low pH, casein is typically in the form of aggregates because the negative charges on the casein are neutralized upon lowering of pH to the isoelectric point of 4.6, with decreased repulsion between the side chains (Chakraborty, A., and S. Basak, J. Photochem. Photobiol. B, 87: 191-199 (2007)). Furthermore, the Ca—P linkages are dissolved, releasing the individual casein and the micellar structure is lost (Gennadios et al. 1994). Treating milk with rennet produces rennet casein which retains the micellar structure. Gelled products such as yogurt and some cheeses are manufactured under low pH conditions. Acid casein may be dried and used in food products or in nonfood applications. With the addition of a base such as Na or Ca(OH)2 to acid casein, at pH in the range from approximately 7 to 9, the casein is solubilized and electrostatic interactions are favored over other interactions such as hydrophobic interactions and hydrogen bonding. The caseinate formed does not have micellar structure. These properties in addition to the random coil structure have been exploited to form edible films and coatings from calcium caseinate (CaCAS), CO2 casein, and sodium caseinate (NaCAS) (Tomasula, P. M., Using dairy ingredients to produce edible films and biodegradable packaging materials, In: Dairy-derived ingredients—Food and nutraceutical uses, M. Corredig, ed., Woodhead Publishing Ltd and CRC Press LLC, Boca Raton, Fla., 2009).
Casein-based edible films have usually been made using a casting process in order to determine their properties. They have excellent tensile and oxygen barrier properties, making them excellent candidates for use in a wide variety of applications (Krochta, J. M., E. A. Baldwin, M. Nisperos-Carriedo, eds, 1994, Edible Coatings and Films to Improve Food Quality, Technomic Publishing Co., Inc., Lancaster, Pa.; Tomasula, 2009). Because of their food-grade status, edible casein films have been proposed for use as part of food systems to prevent migration of components, add to appearance, and to add antimicrobials or nutrients (McHugh, T. H., and J. M. Krochta, Food Technol., January 1994, pp. 97-103). Casein and caseinates have long been used in wet spinning processes for the manufacture of casein fibers for woolen and silk-like fabrics, although they were treated with formaldehyde to harden the fibers (Sutermeister, E., and F. L. Browne, Casein and Its Industrial Applications, Reinhold Publishing Corporation, New York, 1939). Casein fibers have also been proposed for obtaining artificial food protein fibers by spinneret wet spinning (Suckov, V. V., et al., Die Nahrung., 24: 893-897 (1980)).
Recently, electrospinning, a technology for making nonwoven mats from continuous fibers with thicknesses on the nano or microscale, has been used for applications ranging from building tissue engineering scaffolds to use as filter media (Greiner, A., and J. H. Wendorff, Angew. Chem. Int. Ed., 46: 5670-5703 (2007)). The fibers have a high surface area, on the order of 1000× greater than their volume, yielding electrospun products with increased surface efficiency compared to cast films. Electrospinning involves applying a high voltage to a solution containing the polymer. As a solution that is spinnable is discharged dropwise through a nozzle, the electric field causes the drop to form in a cone shape which then forms a continuous jet. The jet becomes narrower and forms an open coil as it approaches a counter electrode. The solvent simultaneously evaporates, precipitating the polymer on the counter electrode. The drop is balanced at the nozzle by its surface tension and is ejected when the electric field is opposed by the solution electrostatic forces that become larger than the surface tension (Greiner and Wendorff, 2007). For successful creation of fibers, electrospinning requires solubility in the solvent, the electric field needs to exceed that of the surface tension at the nozzle to form the cone, and entanglement of the molecular chains of the polymer, which is a function of the viscosity of the solution (Stijnman, A. C., et al., Food Hydrocolloids, 25: 1393-1398 (2011)). Uneven jet formation or electro spraying results in fibers that are interspersed with beads and other shapes. While electrospinning has been successfully applied to synthetic polymers, it has more recently been applied to natural polymers.
There are several examples of electrospinning of natural (non food) and synthetic polymers in non aqueous solvents, but there are relatively few examples of natural polymers electrospun from aqueous solutions, which would include polysaccharides and proteins for food applications. Stinjman et al. (2011) found that the minimum requirements for electrospinning of polysaccharides, such as the conditions under which a jet and then fibers were formed, were shear-thinning behavior at shear rates less than 1000 s−1 and overlap concentration, a measure of the chain to chain interactions and entanglement. Under these conditions, fibers were formed from dextran and pullulan (PUL). Electro spun proteins have required use of a process aid or carrier such as poly(ethylene oxide) (PEO), which has been used to electrospin several proteins, polysaccharides, and cellulose derivatives that cannot be electrospun alone (Alborzi, S., et al., J. Food Sci, 75: C100-107 (2010)). PEO is believed to lower the surface tension and electrical conductivity of the solution, thus enabling electrospinning of the mixture. However, proteins electrospun with PEO are not edible.
Electro spinning of proteins without a carrier has been demonstrated for zein and gelatin. Zein was electrospun from 70% EtOH solutions (Miyoshi, T., et al., Polymer International., 54: 1187-1190 (2005); Kanjanapongkul, K., et al., J. Appl. Poly. Sci., 118: 1821-1829 (2010)) and gelatin was electrospun from water only (Zhang, S., et al., J. Biomedical Materials Research, Part A, 90: 671-679 (2009). Gelatin at a 12.5 wt % concentration (150A, 75B gelatin) was also used as a carrier for electrospinning of proteins such as whey protein isolate, ovalbumin, BSA, soy protein isolate, and NaCAS, with optimal spinning temperature of 40° C. (Nieuwland, M., et al., Innovative Food Sci. and Emerging Tech., 20: 269-275 (2013)). The ability to electrospin a particular protein was also found to be related to an ultrasonic treatment that was required to disrupt aggregated proteins. A harsh treatment of the NaCAS-gelatin solution was required prior to electro spinning.
We have determined the molecular parameters and the operating conditions necessary to electrospin aqueous solutions of proteins (e.g., CaCAS, NaCAS) and polysaccharides (e.g., pullulan) for potential food applications The proteins may also be blended together and fats with milk fats added to form emulsified structures.